Molecular Mechanisms of Immunometabolic
Dysfunction in Multiple Sclerosis
DISSERTATION
zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.)
eingereicht an der
Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin vorgelegt von
M.Sc. Molekularbiologin Aline Tänzer
Präsidentin der Humboldt-Universität zu Berlin Prof. Dr.-Ing. Dr. Sabine Kunst
Dekan der Lebenswissenschaftlichen Fakultät der Humboldt-Universität zu Berlin Prof. Dr. Bernhard Grimm
Gutachter:
1. Prof. Dr. Dirk Brockmann 2. Prof. Dr. Stefan M. Gold 3. Dr. Benedikt Beckmann
II This research project was carried out at the Charité University Hospital Campus Benjamin Franklin, Berlin, in the work group Neuropsychiatry of Prof. Dr. Stefan M. Gold.
III
“According to Darwin’s Origin of Species,
it is not the most intellectual of the species that survives; it is not the strongest that survives;
but the species that survives is the one that is able best to adapt and adjust
to the changing environment in which it finds itself.”
Leon C. Megginson paraphrasing Charles Darwin’s Origin of Species
IV
Abstract
Multiple Sclerosis (MS) is a chronic neurodegenerative disease of the central nervous system characterized by autoimmune-mediated mechanisms. T cells have been associated as central pro-inflammatory mediators in MS pathogenesis. In healthy individuals, immune cells adapt metabolic programs like mitochondrial respiration and glycolysis based on their function and inflammatory phenotype. However, the relevance of metabolic reprogramming and associated pro-inflammatory mechanisms in T cell subpopulations in MS disease is not well understood yet. To address this question, Relapsing Remitting MS (RRMS) patients and meticulously matched healthy control (HC) participants were recruited as part of the clinical study Depression and Immune Function in MS (n=62). Blood samples, after a period of fasting, were collected and CD4+ and CD8+
T cells isolated from peripheral blood mononuclear cells (PBMC). The results obtained demonstrated decreased mitochondrial and glycolytic activity specific to CD4+ T cells in the MS patient cohort compared to the HC participant cohort. Furthermore, increased CPT1a mitochondrial membrane protein levels were detected in CD4+ T cell subpopulations in the MS patient cohort as assessed in comprehensive flow cytometry PBMC phenotype investigations. The analysis of the CD4+ CD25- CD127+ conventional T
cell subpopulation moreover revealed a trend of decreased IL7-Rα expression levels in MS patients. Gene expression measurements of pro-inflammatory and metabolic genes did not reveal alterations in MS patients’ T cell subpopulations. The results obtained in this study allude to dysfunctions in metabolic reprogramming in T cell subpopulations in MS patients and help to better understand the contribution of immunometabolism in the pathogenesis of MS disease.
V
Zusammenfassung
Multiple Sklerose (MS) ist eine chronische neuro-degenerative Erkrankung des zentralen Nervensystems, die durch auto-immun-bedingte Prozesse charakterisiert ist. T Zellen wurden als wesentliche pro-inflammatorische Mediatoren mit der Pathogenese der MS assoziiert. In gesunden Individuen passen Immunzellen ihren Metabolismus, wie die mitochondriale Atmung und Glykolyse, ihrer jeweiligen Funktion und ihrem inflammatorischen Phänotyp an. Im Krankheitsverlauf der MS ist die Bedeutung der metabolischen Anpassung und der damit verbundenen pro-inflammatorischen Mechanismen von T Zell-Subpopulationen noch nicht eindringlich erforscht. Um dieser Fragestellung nachzugehen wurden Relapsing Remitting MS (schubförmig, RRMS) Patienten und sorgfältig aufeinander abgestimmte gesunde Kontrollprobanden als Teil der Studie Depression und Immunfuktion bei MS rekrutiert (n=62). Den Patienten und gesunden Kontrollprobanden wurde Nüchternblut entnommen, woraus periphäre mononukleäre Blutzellen (PBMC) aufgearbeitet wurden, um anschließend CD4+ und CD8+
T Zellen zu isolieren. Die erzielten Ergebnisse zeigten CD4+ T Zell-spezifische Verringerungen der mitochondrialen Atmung und glykolytischen Aktivität in der MS Patienten Kohorte im Vergleich zur Kohorte der gesunden Kontrollprobanden. Darüberhinaus wurden, zusätzlich zu den umfangreichen phänotypischen Charakterisierungen der PBMCs via Durchflußzytometrie, erhöhte Werte des mitochondrialen Membranproteins CPT1a in CD4+ T Zell-Subpopulationen in der MS Patienten Kohorte detektiert. Die Analyse der CD4+ CD25- CD127+ konventionellen T Zell-Subpopulation ergab leicht erniedrigte Werte von IL7-Rα in MS Patienten. Genexpressionsanalysen, die mit pro-inflammatorischen und metabolischen Genen assoziiert sind, ergaben keine Veränderungen in den T Zell-Subpopulationen der MS Patienten. Die in dieser Studie erzielten Ergebnisse weisen auf Funktionsstörungen bei der metabolischen Anpassung in T-Zell-Subpopulationen bei MS Patienten hin und helfen, den Beitrag des Immunmetabolismus bei der Pathogenese der MS Erkrankung besser zu verstehen.
VI
Table of Contents
Abstract ________________________________________________________________ IV Zusammenfassung ________________________________________________________ V 1 INTRODUCTION _______________________________________________________ 1 1.1 Multiple Sclerosis _____________________________________________________ 1 1.1.1 Disease Progression, Symptoms and Risk Factors _________________________ 1 1.1.2 Current Treatment _________________________________________________ 5 1.2 Principals of the Immune System ________________________________________ 5 1.2.1 The Innate Immune System __________________________________________ 6 1.2.1.1 Natural Killer cells ______________________________________________ 7 1.2.1.2 Monocytes ____________________________________________________ 8 1.2.2 The Adaptive Immune System ________________________________________ 9 1.2.2.1 T lymphocytes – development, differentiation and activation ___________ 9 1.2.2.1.1 CD4+ T lymphocytes ________________________________________ 111.2.2.1.2 CD8+ T lymphocytes ________________________________________ 14 1.2.2.2 B lymphocytes - development, differentiation and activation ___________ 15 1.3 Immunometabolism _________________________________________________ 18 1.3.1 Main Cellular Energy Pathways ______________________________________ 18 1.3.1.1 Fatty Acid β-oxidation and the Mitochondrial Membrane Protein CPT1a __ 21 1.3.1.2 Electron Transport Chain and Oxidative Phosphorylation ______________ 22 1.3.1.3 Glycolysis and the Glucose Transporter GLUT1 ______________________ 24 1.3.1.4 Tri-citric Acid Cycle ____________________________________________ 25 1.3.1.5 Pentose Phosphate Pathway _____________________________________ 26 1.3.1.6 Amino Acid and Glutamine Metabolism ____________________________ 26 1.3.2 Regulation of Immune Cells by Adapting Energy Metabolism ______________ 26 1.3.3 HPA-axis Stress Response Signaling ___________________________________ 29 1.3.4 T cell Exhaustion __________________________________________________ 31 1.4 Multiple Sclerosis and Immunometabolism _______________________________ 31 1.5 Aims ______________________________________________________________ 34 2 MATERIALS AND METHODS _____________________________________________ 36 2.1 Clinical Study Depression and Immune Function (DENIM) ___________________ 36 2.1.1 Background and Aim of the Clinical Study DENIM _______________________ 36 2.1.2 DENIM Study Organization and Set-up ________________________________ 36 2.1.2.1 Study Organization ____________________________________________ 36 2.1.2.2 Study Visit Set-up _____________________________________________ 37 2.1.2.3 Inclusion and Exclusion Criteria ___________________________________ 37 2.1.2.4 Telephone Screening for Study Eligibility ___________________________ 38 2.1.2.5 Case Report Forms – Questionnaires, Assessments, Tests ______________ 38 2.1.2.6 Biological Material Collected _____________________________________ 39 2.1.3 DENIM Study Recruitment Period and Outcome ________________________ 40
VII 2.1.3.1 Recruitment Period ____________________________________________ 40 2.1.3.2 Sample Analysis _______________________________________________ 40 2.2 Materials __________________________________________________________ 41 2.2.1 Laboratory Equipment _____________________________________________ 41 2.2.2 Glass and Plastic Equipment ________________________________________ 42 2.2.3 Reagents and Chemicals ___________________________________________ 43 2.2.4 Cell Culture Media ________________________________________________ 44 2.2.5 Materials for RNA isolation and cDNA Synthesis _________________________ 44 2.2.6 Materials and Reagents for qRT-PCR Analyses __________________________ 44 2.2.7 Buffer and Stock Solutions for Magnetically Activated Cell Sorting and
Flow Cytometry Analyses ___________________________________________ 45 2.2.8 Magnetically Activated Cell Sorting and Flow Cytometry Antibodies _________ 46 2.2.9 Software used for analyses _________________________________________ 48 2.3 Methods ___________________________________________________________ 49 2.3.1 Cellular Biology ___________________________________________________ 49 2.3.1.1 Cryo-preservation of Peripheral Blood Mononuclear Cells (PBMC) _______ 49 2.3.1.2 Thawing of PBMCs _____________________________________________ 49 2.3.1.3 MACS Positive Selection ________________________________________ 50 2.3.1.4 Cell Culture of Sorted PBMC subtypes _____________________________ 50 2.3.2 In vitro Metabolic Analyses using the Seahorse XFe96 Analyzer _____________ 51
2.3.2.1 Establishing the Mitochondrial Stress Test Analysis Protocol ___________ 51 2.3.2.2 Metabolic Analyses of CD4+ T cells, CD8+ T cells and non-CD4+/CD8+ T cells 55
2.3.2.3 Evaluation of MS Patient and HC Participant Samples _________________ 56 2.3.3 Flow Cytometry Analyses ___________________________________________ 58 2.3.3.1 Intracellular and Extracellular Staining _____________________________ 58 2.3.3.2 Titration of Antibodies _________________________________________ 59 2.3.3.3 Gating Strategy _______________________________________________ 59 2.3.4 Gene Expression Analyses __________________________________________ 64 2.3.4.1 Preparation and Conservation of RNA _____________________________ 64 2.3.4.2 RNA Isolation and cDNA Synthesis ________________________________ 65 2.3.4.3 qRT-PCR Analyses _____________________________________________ 65 2.3.5 ELISA Analyses of Salivary Cortisol Hormone ___________________________ 66 2.3.6 Statistical Analyses ________________________________________________ 66 3 RESULTS ____________________________________________________________ 67 3.1 MS Patient and Healthy Participant Cohort Characteristics __________________ 67 3.2 In vitro Analysis of Immune Cell Energy Metabolism using the
Seahorse XFe96 Analyzer ______________________________________________ 71 3.2.1 Metabolic Assay Verification ________________________________________ 71 3.2.2 Mitochondrial Energy Metabolism Profile of Purified CD4+ T cells ___________ 72
3.2.3 Mitochondrial Energy Metabolism Profile of Purified CD8+ T cells ___________ 73
3.2.4 Mitochondrial Energy Metabolism Profile of non-CD4+/CD8+ T cells _________ 74 3.2.5 Glycolytic Activity of Purified Immune Cells ____________________________ 76 3.2.6 Metabolic Phenotype of Purified Immune Cells _________________________ 77
VIII 3.3 Flow Cytometry Analyses _____________________________________________ 79 3.3.1 Phenotypic Characterization of Immune Cell Subpopulations ______________ 79 3.3.2 CPT1a Expression in Immune Cell Subpopulations _______________________ 81 3.3.3 PD-1 Expression in Immune Cell Subpopulations ________________________ 84 3.3.4 IL7-Rα and IL2-Rα expression in conventional T cells _____________________ 85 3.4 mRNA Gene Expression in CD4+ T cells, CD8+ T cells and non-CD4+/CD8+ T cells __ 86
3.4.1 mRNA Analysis of Pro-Inflammatory and Energy Metabolism Genes TNF-a, NFκB1, NFκB3, GLUT1 and CPT1a __________________________________________ 87 3.4.2 mRNA Analysis of Glucocorticoid Receptor (GR) and Glucocorticoid-induced Leucine Zipper (GILZ) ___________________________________________________ 88 3.5 Cortisol Levels in Saliva Samples _______________________________________ 89 3.6 Clinical Data Correlation Analyses ______________________________________ 90 4 DISCUSSION _________________________________________________________ 92 4.1 The DENIM Study Provides a Robust MS Patient and HC Participant Cohort ______ 92 4.2 Detection of Comparable Phenotyping Profiles of Major T cell Subpopulations in MS Patients and HC Participants ______________________________________ 93 4.3 Decreased Immunometabolic Potential is specific to CD4+ T cells from MS Patients ________________________________________________________ 96 4.4 CD4+ and CD8+ T cell Subpopulations from MS Patients Express Increased
Levels of the Mitochondrial Membrane Protein CPT1a _____________________ 100 4.5 HPA Axis Activity Corresponds between MS Patients and HC Participants
While GR and GILZ Gene Expression is Altered in CD4+ and CD8+ T cells ________ 106
4.6 B cells and NK cells show Modified Phenotype Profiles and CPT1a Protein
Levels in MS Patients versus HC Participants _____________________________ 108 Limitations, Strengths, Outlook ____________________________________________ 112 List of Abbreviations ____________________________________________________ 114 References ____________________________________________________________ 117 Figure Index ___________________________________________________________ 133 Table Index ____________________________________________________________ 135 Supplements ___________________________________________________________ 136 Appendix ______________________________________________________________ 137 Flow Cytometry Analyses ____________________________________________ 137 DENIM Study Flyer _________________________________________________ 140 DENIM Study Case Report Form _______________________________________ 142 Acknowledgements _____________________________________________________ 198 Peer-reviewed Publications _______________________________________________ 199 Eigenständigkeitserklärung ______________________________________________ 200
1
1
Introduction
1.1
Multiple Sclerosis
Multiple Sclerosis (MS) is a chronic neurodegenerative disease of the central nervous system (CNS) characterized by the demyelination of neurons believed to be induced by autoimmune mechanisms. Worldwide, approximately 2.5 million people are diagnosed with MS and about 200 000 people in Germany.
Disease influencing factors include environmental and genetic components leading to a broad and heterogeneous clinical presentation. To date, magnetic resonance imaging (MRI) as well as cerebral spinal fluid (CSF) analyses provide the basis for the clinical diagnosis, characterization and progression of MS and contribute to the understanding of the disease.
1.1.1 Disease Progression, Symptoms and Risk Factors
MS is characterized by a chronic state of neuroinflammation of the CNS, which progresses over time. The average age of disease onset is 30 years and due to increasing physical impairments, considering current treatment, approximately 50% of patients require a wheelchair 25 years after diagnosis [1]. The course of disease and disability progression depends on the kind of MS.
Disease development can generally be characterized by three stages: 1) a pre-clinical, 2) a relapsing-remitting MS (RRMS) and 3) a progressive stage. Figure 1 shows the course of disease with an overall increasing disease burden including clinical disability and MRI activity as well as decreasing brain volume over time. The pre-clinical stage oftentimes goes unrecognized unless MRI scans incidentally discover typical neuronal inflammatory regions, defined as brain lesions. This stage of MS is mostly asymptomatic and defined as the radiologically isolated syndrome (RIS). Following the RIS, usually the clinically isolated syndrome (CIS) manifests itself and is followed by the RRMS stage. Now, in CIS and RRMS, patients show distinct clinical symptoms, most prominently optical neuritis, sensory deficits and motor dysfunctions. As relapses increase over time, symptoms accumulate and disability worsens. In the RRMS stage, relapses with neurological dysfunction and
2 neuronal inflammation are redeemed by phases of remission during which symptoms decline or, especially in the early phases, subside to a great extent. However, with accumulating relapses, the disease burden increases, brain volume decreases and patients enter the third clinical stage, progressive MS. At this stage, neurological dysfunction progressively worsens without phases of remission occurring. Clinical manifestation of progressive MS following RRMS is termed secondary progressive MS (SPMS). If the disease onset is progressive from onset on, however, it follows the course of primary progressive MS (PPMS) (Figure 1).
Figure 1: Stages and disease progression in Multiple Sclerosis. The course of MS shows an overall increase of disease burden accompanied by an increased clinical disability and decreasing brain volume over time. The pre-clinical stage includes the radiological isolated syndrome (RIS), which is often asymptomatic and incidentally discovered on MRI scans. RIS develops into the clinical stage, clinically isolated syndrome (CIS). CIS is followed by a relapsing remitting course of disease (RRMS) during which symptoms with periods of relapses and neurological dysfunction as well as pro-inflammation manifest and get replaced by periods of remission. The final clinical stage is secondary progressive MS (SPMS), which evolves from RRMS and lacks remission phases. If disease onset progresses from the beginning, it is termed primary progressive MS (PPMS). Brain volume and atrophy is measured by Magnetic Resonance Imaging (MRI) scans as a biomarker for MS providing information about newly occurring, active and persistent brain lesions and with that disease progression over time. (tailored based on [2])
To date, MRI scans of the CNS are the most common biomarker for MS disease activity [2]. Using MRI scans, active, newly occurring and persistent lesions can be detected,
3 visualized and observed over time allowing the measurement of disease burden and progression. CNS lesions and inflammation are a hallmark of MS and are accompanied by a blood brain barrier (BBB) leakage allowing the scarcely restricted infiltration of immune cells from the peripheral blood stream [1]. One of the first cell populations found in lesions inside the CNS include lymphocytes [3]. Therefore, MS is presumed to be of autoimmune etiology initiated by over- and auto-reactive T lymphocytes finally targeting myelin sheaths of neurons. This process of BBB leakage and CNS infiltration has been described especially in RRMS patients and leads to the diverse clinical symptoms.
The main symptoms patients present with include muscle weakness, fatigue, impairments in motor function and coordination, cognitive impairments and major depressive disorder (MDD). MDD is the most prevalent psychiatric comorbidity in MS patients with a lifetime prevalence of developing depression of up to 50% [4]. Studies have shown strong associations between an increased inflammatory profile in MS patients with increased fatigue and depression scores further contributing to the inflammatory processes observed in MS patients [5]–[7]. Importantly, symptoms can be very diverse and depend on the degree of disability and overall disease burden of a patient.
Causes for the onset of MS are multifaceted and include genetic and environmental factors, sex differences and hormone regulation (comprehensively reviewed in [8]). While the kind of MS and disease severity do not show a genetic component, with first-degree relatives having a 2-5% higher risk for developing MS, disease susceptibility seems to relate to genetic effects. Numerous studies showed that most genes associated with MS seem to relate to either the innate or adaptive immune system. One of the most dominant risk factors is the HLA-DRB1*15:01 allele in the class IImajor histocompatibility complex (MHC) gene [9]. Examples for non-MHC associated MS-susceptibility loci are the
interleukin-7 receptor α (IL7-Rα or CD127) and interleukin-2 receptor α (IL2-Rαor CD25) genes. Genome wide association studies (GWAS) detected small nuclear polymorphisms (SNP) in the IL7Rα gene in MS patients, where the SNP most likely induces an increased
abundance of soluble IL7-Rα protein [10]. The IL7-Rα is essential in T and B lymphocyte maturation, development and homeostasis and downstream activation of the janus kinase (JAK) and signal transducer activators of transcription (STAT) pathways- both involved in gene transcriptions following immune responses and in direct interaction with
4 the nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) pathway [11]. The IL2-Rα is upregulated on activated T cells and therefore essential in cellular proliferation, survival and T cell homeostasis [12],[13]. Taken together, genetic risk factors for MS hint toward strong associations with immune regulation, however, no single genes have been defined to be involved in causing MS.
Another contributor to MS development and progression are environmental influences. One of the most widely discussed environmental factor in MS disease is the protective effect of ultra violet (UV) radiation exposure. Especially early in life before the age of 20, elevated UV radiation exposure and with that increased vitamin D levels have been shown to reduce MS-risk later in life [14]. Vitamin D signaling greatly affects anti-inflammatory immune cell pathways via T and B lymphocyte suppression and regulatory T cell (T regs) induction, with that promoting protective immune tolerogenic effects [15]. Vitamin D levels have also been shown to have protective effects in other autoimmune diseases like type 1 diabetes (T1D), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [16], indicating it being an essential influence in immunity.
The effects of sex differences and hormones have also been studied extensively in MS. The 2.5:1 ratio of females:males affected by MS is striking. This ratio has been explained with parent of origin effects where, for example disease associations like the risk allele HLA-DRB1*15:01 show a stronger association with females [17]. Additionally, X- and Y-chromosome-linked genes have been shown to contribute to gene-dosage effects and immune regulation of innate and adaptive immune responses [18]. Furthermore, hormone regulation greatly affects cells of the innate and adaptive immune system displaying distinct differences and adaptations in females and males (particularly estrogen and testosterone, respectively). This effect is most prominent, considering the strong protective effect of pregnancy from relapses [19].
Taken together, genetics, environmental influences, sex and hormones allow a deeper understanding of MS disease and help to better comprehend the underlying causes leading to the complex pathogenesis.
5
1.1.2 Current Treatment
Treatment for MS includes medications to treat occurring symptoms as well as basic immune-modulatory medication. Symptom management aims to increase the quality of life and reduce MS symptoms. Especially during relapses, patients oftentimes are treated with cortisone to manage peripheral and CNS inflammation.
Current immune-modulatory medications to treat RRMS are targeted at innate and adaptive immune cells. They suppress pro-inflammatory signaling and have become more advanced in the last decade with to date more than ten drugs approved by the American Food and Drug Administration (FDA). Baecher-Allan and colleagues most recently published an extensive review summarizing current drugs, their specific targets and the mechanisms of action [2]. In summary, the modes of action include the trapping of T lymphocytes inside the lymph nodes and with that prevention of T lymphocyte migration via the BBB into the CNS, the promotion of T regs and decrease of MS-disease inducing Th1/Th17 cells as well as the inhibition and depletion of B lymphocytes. Considering efficacy, side effects, relapse history and CNS lesions, the clinician and patient decide which drugs to use. The efficacy of the drug is measured by the reduction in relapses and a decrease of inflammation. Using MRI scans, disease progression and brain atrophy is determined [20].
Almost all of the FDA approved drugs target the relapsing inflammatory stage and are largely not effective in the progressive forms of MS. Furthermore, to date, medications are effective in limiting relapses, but do not fully prevent disease progression.
Broadening the understanding of the molecular mechanisms of immune activity in MS may allow a deeper understanding of causative mechanisms underlying the disease.
1.2
Principals of the Immune System
The human immune system is composed of an enormous multitude of mechanisms all prepared to target threats to the body and fight infections. It entails molecular structures, cells, tissues and organs whose main function is the elimination of pathogenic agents as well as aged or cancerous body cells.
6 The immune response requires the regulation and interaction with other body systems, like the central nervous system and (neuro-) endocrine mechanisms as well as energy demand and response signaling pathways. It is classified into innate and adaptive immunity, which constantly interact to initiate effective defense mechanisms involving humoral (non-cellular) and cellular structures. The cellular components of the blood arise from pluripotent hematopoetic stem cells (HSC) in the bone marrow that possess the ability to develop into common lymphoid progenitor cells (T and B lymphocytes and natural killer cells) as well as common myeloid progenitor cells (monocytes, dendritic cells, granulocytes). Peripheral blood mononuclear cells (PBMCs) are composed of about 25-60% CD4+ T lymphocytes, 5-30% CD8+ lymphocytes, 5-10% B lymphocytes, 10-30% NK
cells, 5-10% monocytes and 1-2% dendritic cell [21]. The various cell types and their functions within innate and adaptive immunity are subsequently discussed in detail.
1.2.1 The Innate Immune System
Innate immune response mechanisms are coded in the germline and compromise the first line of defense against pathogens like bacteria, viruses, worms or fungi. Examples for innate immune cells are neutrophils, basophils, eosinophils (all granulocytes), natural killer (NK) cells and monocytes. They circulate within the peripheral blood stream and constantly monitor for invading pathogens. Humoral components include the complement system, cytokines, chemokines and pattern recognition receptors (PRRs). Cytokines are small proteins involved in cell-specific signaling and communication. A subtype of cytokines are chemokines, which possess the ability to recruit cells to the site of inflammation. Both, cytokines and chemokines can be secreted by cells and bind to their specific cytokine and chemokine cell surface receptors. PRRs can also be membrane bound or soluble, recognizing conserved pathogen associated molecular patters (PAMPs). Examples for highly conserved PRRs are toll like receptors (TLRs). To date, more than 13 TLRs have been identified [22], each able to recognize one or multiple ligands, e.g. microbial motifs and debris from necrotic cells.
The recognition of PAMPs induces an immediate immune response. The release of cytotoxic molecules by innate immune cells induces the killing of extracellular pathogens or apoptosis (programmed cell death) of infected cells. Chemokines and cytokines recruit
7 additional immune cells causing further pro-inflammatory responses and pathogen clearance.
Additionally, most innate immune cells function as antigen presenting cells (APCs). Following the recognition by their TLR, APCs can present processed antigen via their MHC class II for antigen recognition by T lymphocytes of the adaptive immune system. Furthermore, MHC-II presentation can also occur after phagocytosis, the engulfment of cells or particles, of pathogens or infected cells. The interaction of innate and adaptive immune cells and their humoral components is a central element of immune response mechanisms.
Phagocytosis and antigen processing as well as the release of cytotoxic molecules are two central elements in innate immunity. NK cells and monocytes are two cell subpopulations involved in these processes and are subsequently described in more detail.
1.2.1.1 Natural Killer cells
NK cells are granular lymphocytes of the innate immune system essential in anti-bacterial and anti-viral immune responses [23]. They predominantly detect cells that lack MHC class I expression and with that the identification of a cell as self. The main functions of NK cells include the secretion of immune activating cytokines and chemokines and the killing of infected or transformed cells via pathways including perforin/granzyme or death receptor-related pathways [24]–[26]. They express TLRs involved in the recognition of bacterial and viral PAMPs (e.g. TLR2, TLR5, TLR7/8, TLR9) [22] and also play a central role in tumor immunity [25] and human pregnancy [27].
The activation of NK cells by TLR ligands is accompanied by the production of interferon γ (INFγ) and granulocyte-monocyte colony stimulating factor (GM-CSF) and requires the presence of assessor cytokines like IL-2 and IL-12, IL-2 and IL-18 or IL-15 and IL-18 [28]. Consequently, NK cell activation is coupled to assessor processes safeguarding over-reactivity.
Based on their function, cytokine secretion profile and cell surface receptor expression, NK cells are subdivided into regulatory and cytotoxic subtypes. Regulatory NK cells are characterized as CD56+ CD16-/low and are mainly found in lymph nodes, lacking cytotoxic
8 potential. Cytotoxic NK cells are defined as CD56+ CD16+/high, are more differentiated and have the potential to mediate cytotoxicity [29].
INFγ is the signature cytokine of CD56bright NK cells and known to shape Th1-mediated adaptive immunity [26] as well as to induce macrophages to kill intracellular pathogens and activate APCs to up-regulate MHC class I molecules [30]. Additionally, INFγ secretion by NK cells has anti-proliferative effects on cells that have been transformed due to viral infection or malignancies [31].
Taken together, NK cells play an important role in innate immune response and cellular activation as well as in the induction of adaptive immune mechanisms.
1.2.1.2 Monocytes
Monocytes are central APCs of the innate immune system. They get released into the periphery where three monocyte subpopulations have been characterized based on their cell surface receptor expression: classical monocytes (CD14++ CD16-), intermediate
monocytes (CD14++ CD16+) and non-classical monocytes (CD14+ CD16++) [32]. After circulating in the blood, they reach tissue where they differentiate and further mature into tissue resident dendritic cells (DC) or macrophages.
The main functions of monocytes include pro-inflammatory cytokine production following the recognition and phagocytosis of pathogens and the antigen presentation via MHC class I and II molecules. The signature cytokines released include TNFα, IL-1 and IL-6 [33]. Monocytes express numerous TLRs (e.g. TLR2, TLR9) for antigen recognition [34],[35] and phagocytosis. The latter takes place by direct binding to pathogens via PAMP recognition or by detection of antibody- or complement-coated pathogens (opsonization). Unlike NK cells, monocytes do not require the presence of assessor cytokines for their activation. Once they are activated, they release pro-inflammatory cytokines and with that induce the transmigration of other innate and adaptive immune cells to the site of inflammation. Depending on the intruding pathogen, monocytes have been shown to be able to induce helper T cell mediated immune responses and with that the activation of the adaptive immune system [33]. Moreover, monocytes have been shown to act as suppressor cells inducing Treg development and inhibiting T lymphocyte proliferation [36].
9 Taken together, monocytes display an essential role in rapid innate immune response mechanisms and are key components in adaptive immune cell activation, proliferation and suppression.
1.2.2 The Adaptive Immune System
The adaptive immune system compromises defense mechanisms that are acquired, develop from the time of birth and evolve over the course of a lifetime. As described for innate immune responses, adaptive immunity also entails cellular and humoral components. However, contrary to innate immunity, adaptive immunity is highly antigen-specific. Main cell types constituting adaptive immunity include T and B lymphocytes. T lymphocyte subpopulations include helper and cytotoxic T lymphocytes with specific cell surface receptor expression and antigen-specific functions. B lymphocytes produce antigen-specific antibodies, which are part of the humoral structures in adaptive immunity. Furthermore, both, T and B lymphocytes, possess the ability to form memory cells after antigen clearance, a crucial process that allows an immediate immune response during a secondary infection with the same antigen.
The ability of specialized cell subpopulations to respond to specific antigens and generate immune memory are key characteristics of adaptive immunity and are subsequently described in more detail.
1.2.2.1 T lymphocytes – development, differentiation and activation
T lymphocytes originate from stem cells in the bone marrow. Once released, they travel to the thymus (thymocytes) and mature into functional CD4+ and CD8+ T lymphocytes
(subsequently termed CD4+ and CD8+ T cells). In the process of thymic maturation, a
broad repertoire of antigen-specific T cell receptors (TCR) is established.
Most TCRs entail an α- and a β-chain (>85% of T cells) and only the remaining fraction is made up of γ:δ-TCRs. The TCR protein structure comprises a constant unmodified region and a variable region. The variable region is the site of antigen-recognition and specific for each T cell. During T cell development, the α and βgenes of the variable region of the
variable-10
diversity-joining (V-D-J, β-chain) gene segments. This process allows the tremendous diversity of the TCR antigen-recognition repertoire, which is estimated with approximately 1011 specificities [37].
The TCR is part of a group of a membrane-bound CD3-protein complex, whose interaction is required during antigen recognition. The CD3-complex is made up of a γ and δ chain as well as two ε and two ζ chains. Additionally, T cell co-receptors are required for antigen-recognition and T cell activation, including CD40L (CD154), CD28 and CD4 (on CD4+ T cell subpopulations) or CD8 (on CD8+ T cell subpopulations) [38].
During thymic maturation, thymocytes are selected based on their ability to recognize self and foreign antigen via their TCR. Briefly, during the first major stage of thymic T cell differentiation, double-negative CD4- CD8- T cells undergo TCR rearrangement. This
allows them to progress to the second major stage of differentiation where they are termed double-positive T cells expressing both CD4and CD8 co-receptors. At this point, APCs present a multitude of antigens and self-peptides via MHC classes I and II to the premature T cells. Now, positive and negative selection occurs during which highly self-reactive T cells (binding to self-antigens on the MHCs) and T cells that do not bind MHC are eliminated. T cells that are weakly reacting to self-peptides receive survival signals allowing them to mature into the third stage of differentiation. During this stage, either the CD4 or CD8 receptor is down-regulated yielding single-positive (SP) naïve CD4+ CD8- T
cells and SP naïve CD4- CD8+ T cells. They emigrate from the thymus into the blood stream
as well as secondary lymphoid organs (like the spleen, Peyer’s patches in the gut, mucosal tissue, tonsils or adenoids). Their main function is immune surveillance. Once they encounter their specific antigen, they mature into functional helper or effector T cells. T cell activation requires the binding of the antigen-specific TCR-complex to the MHC-antigen complex on the cell surface of APCs. The MHC-complex is composed of the co-stimulatory receptors CD80 or CD86 (binding to CD28 on the TCR) as well as CD40 (binding to CD40 L on the TCR). CD4+ T cells recognize antigens from pathogens like extracellular bacteria, worms or toxins that have been processed by APCs and presented on their MHC class II. In contrast, CD8+ T cells recognize antigens from intracellular bacteria, viruses and endogenous molecules like tumor proteins that have been processed by APCs and presented on their MHC class I.
11 Taken together, the activation of T cells requires the binding of the TCR-complex to the MHC-antigen complex including all co-receptors. T cell activation induces a cytokine and chemokine environment (also involving other immune cells), which results in the differentiation into specific CD4+ helper T cell subsets and CD8+ effector T cells as well as the recruitment of other immune cells to the site of inflammation.
Following an antigen encounter, memory T cells are formed. They possess the ability to undergo clonal expansion, proliferation as well as activation of other immune cells inducing a fast immune response to a previously intruding antigen. Three memory T cell subsets are classified based on their function and expression of surface receptors: central memory T cells (TCM), effector memory T cells (TEM) and terminally differentiated effector
memory cells re- expressing CD45RA (TEMRA) (summarized in Table 1). Upon activation and
differentiation to memory T cells, CCR7+ naïve T cells (T
N) down-regulate the CD45RA
isoform and express the CD45RO isoform. TCM are CCR7+, have high proliferative
capacities and lower effector functions. TEM are CCR7-, resident in peripheral tissues and
possess the ability to quickly adapt a CD4+ effector or CD8+ cytotoxic T cell phenotype.
The third memory subtype are TEMRA, which are CCR7-, show low proliferative and
functional capacities and characteristics of senescent cells [39].
Taken together, CD4+ and CD8+ memory T cell subpopulations monitor and patrol the
body for intruding pathogens in tissues and the periphery. They are highly effective and time-efficient during a secondary immune response to a previously targeted pathogen in the primary immune response. This is the essence of adaptive immunity allowing pathogen-specific immune responses.
1.2.2.1.1 CD4+ T lymphocytes
Upon activation, CD4+ T
N differentiate into effector T cells including helper T cell (Th)
types Th1, Th2, Th17 and Th1/17. Helper T cells mediate cellular and humoral immune responses specific to the intruding pathogen. They are characterized based on their chemokine receptor and cytokine profile as well as individual transcription factors (Table 1 provides a summary). An additional CD4+ T cell subpopulation are regulatory T cells
12 (Treg), which complement immune activation and are essential for immune homeostasis and self-tolerance.
Th1 cells produce IFNγ and are activated and induced by interleukin (IL)-12 as well as the transcription factors T-bet, STAT1 and STAT4 [40]. Th1 cells are characterized based on the chemokine receptors CXC-chemokine receptor (CXCR) 3 and chemokine receptor (CCR) 5 [41]. A Th1-mediated immune response leads to the targeted reaction to intracellular bacteria and the activation of infected macrophages within the inflammatory micro-environment.
Th2 cell development is promoted by IL-4 as well as the transcription factors STAT-6 and GATA-3 [42]. Characteristic transcription factors are CCR4 and Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2) [43]. They secrete IL-4, IL-5, IL-9 and IL-13 and stimulate histamine- and heparin-secreting mast cells contributing to the elimination of extracellular pathogens like bacteria or worms. Furthermore, Th2 cell stimulation leads to the activation of B lymphocytes inducing antigen-specific antibody production.
Th17 cells induce a strong pro-inflammatory immune response to extracellular pathogens like bacteria and fungi. They are characterized based on CCR4 and CCR6 chemokine expression [41]. Cytokines promoting the induction of Th17 cells include IL-6, IL-17, IL-23 and transforming growth factor-β (TGF-β), which also induce the expression of the Th17-specific transcription factors retinoic acid receptor-related orphan nuclear receptor (ROR) γt, RORα and STAT-3 and vice versa [42]. Following pathogen intrusion, Th17 cells induce neutrophil activation, which promotes a fast line of defense via the release of anti-microbials, the elimination of pathogens by phagocytosis and the induction of chemotaxis. Th17 immune response mechanism can be inhibited by Th2 cells, thereby preventing an overreaction of the immune system and with that a damage of self-tissue. Th1/17 cells have been described as a transitional state between Th17 and Th1 cells producing IFNγ and IL-17 [41]. The conversion of Th1 to Th17 cells has been shown to impact both, MS disease progression as well as regulatory functions [44].
T regs are classified as Forkhead box P3+ (FoxP3) CD25+ CD4+ T cells. They are primarily generated during thymic selection (referred to as tT regs or natural T regs) and possess a relatively high affinity to self-antigens on the border to being negatively selected [45].
13 Additionally, peripheral T regs (pT regs) can be generated extrathymically and induced T regs (iT regs) can be generated in cell culture stimulated by transforming growth factor-β (TGF-β). The transcription factor FoxP3 is the master regulator of T regs and along with CTLA-4 essential for T reg functionality. Importantly, T regs express no or only low levels of IL-7 (CD127), while CD4+ CD25- CD127+ conventional T cells (all CD4+ T cells but T regs) express IL-7Rα on their surface [46]. IL-10, IL-35 and TGF-β are the signature cytokines secreted by T regs and induce T reg activation and proliferation. T regs are crucial for the suppression of excessive immune responses as well as the maintenance of immunological unresponsiveness to self-antigens.
Table 1 provides a comprehensive overview of the CD4+ T helper cell and memory T cell
subtypes including the chemokine receptor expression profiles characteristic for each phenotype. It summarizes the previously described subsets and their descriptive transcription factors, cytokine profiles, as well as natural functions.
14
Table 1: CD4+ helper T cell and memory cell subtypes, transcription factors, cytokine
profiles, phenotype and natural function. Modified based on[39],[43],[47].
T cell
subset TF inducing cytokines secreted cytokines pheno-type natural function Th1 T-bet IL-12 IFNγ,
GM-CSF
CXCR3+
CCR5+
cell-mediated immunity, intracellular pathogens Th2 GATA3 IL-4 IL-4, IL-5,
IL-10, IL-13 CCR4
+
CRTh2+ humoral immunity, extracellular parasites
Th17 RORγt IL-1β, IL-6, IL-23, TGF-β
IL-17,
GM-CSF CCR6
+
CCR4+ mucosal immunity, extracellular fungi and
bacteria
Tregs FoxP3 - TGF-β CD25+,
CD127- immune homeostasis, (self-)tolerance
TN IL-2 CD45RA+
CCR7+ progenitor cells, immune surveillance
TCM IL-2, IL-21 CD45RO+
CCR7+ immune memory, B cell help, secondary expansion
TEM IL-4, IL-5,
IL-17, TNF-α, IFNγ
CD45RO+
CCR7- immune memory, B cell help, protection in tissues
TEMRA CD45RA+
CCR7- immune memory, terminally differentiated cells
IL: interleukin, TF: transcription factor, Th: helper T cell, IFN-γ: interferon γ, GM-CSF: granulocyte-macrophage colony-stimulating factor, mTOR: mechanistic target of rapamycin, ERR: estrogen receptor, HIF-1α: hypoxia-inducible factor-1α, FoxP3: forkhead P3, TGF: transforming growth factor, AMPK: AMP-activated protein kinase, TN: naïve T
cells, CM: central memory, EM: effector memory, TEMRA: terminally differentiated effector
memory cells re- expressing CD45RA, TNF-α: tumor necrosis factor-α, CCR: Chemokine Receptor, CXC: CXC-Chemokine Receptor, CRTh2: Chemoattractant receptor-homologous molecule expressed on Th2 cells, RORγt: retinoic acid receptor-related orphan nuclear receptor γt.
1.2.2.1.2 CD8+ T lymphocytes
CD8+ T cells are a T lymphocyte subpopulation that, in contrast to CD4+ T cells, recognize
MHC class I molecules on APCs via their CD8 TCR complex and are very potent pro-inflammatory immune activators (described in 1.2.2.1). They differentiate and proliferate into cytotoxic CD8+ T cells inducing a cascade of pro-inflammatory signaling to fight off
15 the intruding pathogen or neoplastic cells. Signature cytokines of cytotoxic T cells include IL-2, TNF-α and INF-γ. Additionally, due to their function in clearing intracellular viruses, bacteria and cancer cells, CD8+ T cells secrete perforin, granzyme and granulolysin inducing a strong immune attack. To prevent an immune overreaction, cytotoxic T cells require at least three signals to become activated: TCR specific antigen recognition, co-receptor binding (e.g. CD28) and 2 signaling (which may include Th cells that provide IL-2) [48].
To date, three main CD8+ cytotoxic T cell subpopulations have been described. They are differentiated based on their chemokine expression and are unique to their locations in the body. CCR4, CCR6 and CXCR3 are used for their characterization. The specific immune functions of the subpopulations have been studied to a greater extend, but still remain somewhat elusive. CD8+ CCR4+ CCR6- CXCR3- T cells are predominantly found in the
epithelium and lung [49] and are involved in Th2-mediated immunity [50]. CD8+ CCR6+ CCR4- CXCR3- T cells are mainly located in the gut and have been shown to be essential in
Th17-mediated immune responses, while CD8+ CXCR3+ CCR4- CCR6- T cells display key
immune functions in Th1 immunity [50].
CD8+ cytotoxic T cells act on pathogen infected cells or neoplastic cells by releasing
granzyme, granulolysin and perforin. These factors are released from granules within the cytoplasm of cytotoxic T cells and are exerted onto the target cells. They induce apoptosis, form holes in the target cell membranes, inhibit virus replication and act as anti-microbials by creating a pro-inflammatory micro-environment.
1.2.2.2 B lymphocytes - development, differentiation and activation
Just like T cells, B lymphocytes (subsequently termed B cells) are lymphocytes derived from pluripotent hematopoetic stem cells in the bone marrow and are part of the adaptive immune system. B cells and the immunoglobulins (Ig, also known as antibodies) they produce make up an essential part of humoral immunity protecting against an enormous variety of pathogens. The B cell antibody repertoire entails antibodies that can recognize more than 5x1013 specificities [51]. B cells can produce five different types of immunoglobulins: IgM, Ig,A, IgG, IgD and IgE. All of them differ in size, specificity and functionality [51].
16 The protein structure (variable and constant regions) as well as the genetic mechanisms underlying the production of antibodies resemble that of the TCR on T cells. Briefly, during cellular development in the bone marrow, B cells undergo the following stages: stem cell, early pro-B cell, late pro-B cell, large pro-B cell, small pre-B cell and immature B cell [52]. When the precursor forms of the B cells undergo these stages, rearrangements of the variable (V), joining (J) and diversity (D) regions of the heavy chain and VJ rearrangement of the light chain occurs. Heavy-chain rearrangement occurs after the early pro-B cell stage and its success is tested selecting for functional heavy chains. If the cell passes that first functionality checkpoint, it undergoes light-chain rearrangement followed by a second checkpoint selecting for functional light chains. If that succeeds, the cell moves on to the immature B cell stage.
Following these briefly summarized developmental stages, the immature B cell possesses an antigen-specific immunoglobulin (Ig), which is embedded into the cell membrane and part of the B cell receptor (BCR) complex. Immunoreceptor tyrosine-based activation motifs (ITAMS) are linked to Ig on the BCR and induce activation signaling upon antigen-specific immunoglobulin binding [53].
Immature B cells that recognize self-antigens in the bone marrow are eliminated, rescued or become tolerant. The process of central tolerance includes BCR editing, clonal elimination, depletion, inactivation or anergy. Immature B cells that do not recognize self-antigens in the bone marrow migrate into the lymphatic system as immature transitional B cells where they undergo the process of peripheral tolerance after encountering their BCR specific antigen. They either become anergic, undergo clonal deletion or move on to mature. BCR-specific antigen recognition as well as IgM and IgD expression characterizes mature naïve B cells. Mature naïve B cells are further characterized as CD20+ CD3
-lymphocytes [54] and have been described to secrete the immunosuppressive cytokine IL-10 [55]. CD20 is expressed from the pre-B cell stage until the mature naïve as well as memory B cell stage, however, plasma cells lack CD20. Mature naïve B cells circulate between lymphoid vessels and the blood stream where they encounter APCs as well as helper T cells and patrol for intruding pathogenic antigens.
Interestingly, mature B cells undergo a process termed somatic hypermutation. In addition to immunoglobulin gene rearrangement in the BM, this highlights a second step in B cell antigen recognition diversification. During somatic hypermutation,
high-17 frequency point mutations occur at the hypervariable sites of the variable regions on the light and heavy chains. These sites are the points of contact with antigens. Therefore, the affinity of the antibody to an antigen can get increased or decreased allowing antibodies with medium or low antigen affinities to become highly-specific for pathogenic antigens [51]. Somatic hypermutation is specific to B cells and has not been described in T cells. Compared to T cells, B cells do not depend on MHC presentation for activation. B cells can recognize and bind antigen directly via their BCR along with co-receptors and have the ability to bind non-protein antigens like foreign polysaccharides and DNA independent of T cell engagement [56]. Nevertheless, B cells can bind antigens by their antigen-specific BCR. The BCR-antigen complex gets engulfed and the foreign antigen is processed and subsequently presented on the cell surface via MHC class II for recognition by e.g. helper or memory T cells. B cells are essential APCs for helper T cells and initiate helper T cell activation, expansion as well as differentiation. Similarly, B cells may depend on helper T cell-mediated initiation of activation, proliferation and differentiation following antigen presentation by T cells [57]. Once the B cell recognizes its specific antigen, the cell undergoes clonal expansion and develops into an antibody-producing plasma cell fighting off the pathogen and activating additional immune mechanisms and cells of the innate and adaptive immune system.
In addition to B cells undergoing clonal expansion, antigen-specific memory B cells are formed, which able to effectively and efficiently fight re-occurring antigens [58]. These long-lived memory B cells are CD20+ and able to persist for years [59]. They have been
described to be CD27+ and produce pro-inflammatory cytokines like IL-12, lympho toxin α (LTα) and TNF-α [55].
In summary, B cells are very potent antigen-specific lymphocytes with a tremendous antigen recognition repertoire that is vital in complementing T cell immune responses. The described developmental stages and numerous functions of B cells require the fine interaction and modulation of cellular energy pathways. Here, the ability to switch between low and high energetic states is fundamental and allows successful immune cell functionality. The following text discusses the central role of metabolism in immune cell function.
18
1.3
Immunometabolism
The word metabolism is derived from the Greek word μεταβολισμός (metavolismós), which means change. Immune cells have numerous specifications, functionalities and responsibilities and with that need to display very distinct metabolic profiles to meet their individual tasks and fulfill their target functions. Therefore, the ability to adapt and change their metabolic profile is crucial for healthy immune response mechanisms and host survival.
One of the first researchers analyzing cellular metabolic function was Otto Warburg. In 1956, he described that even under aerobic conditions cancer cells heavily depend on glycolysis instead of oxidative phosphorylation (OXPHOS) to meet their increased energy demand- an observation that has subsequently been termed the Warburg Effect [60],[61]. From there on, the analysis of cell-specific energy pathways has spread to all kinds of research areas including immunology.
Subsequently, the main cellular energy generating pathways are described and linked to immune cell function.
1.3.1 Main Cellular Energy Pathways
Cells are regulated by extrinsic and intrinsic mechanisms and therefore modulate their specific activities and metabolic pathways based on their requirements for survival, growth or development. The ability to use and switch between different pathways to generate energy from nutrients like sugars, proteins and fats are key characteristics of cellular energy metabolism. Typically, the energy is stored in the chemical bonds of these nutrients and the catabolism (break down) of these bonds releases energy, which is used to generate energy rich molecules like adenosine triphosphate (ATP) that the cell can subsequently use.
Importantly, cellular chemical reactions have exceptionally high reactive potential and are therefore always profoundly controlled for. Energy signaling pathways in eukaryotic cells, including human immune cells, occur either in the cellular cytosol or within mitochondria. The cytosol is defined as the liquid inside the cell and is surrounded by the cell membrane. Glycolysis, the pentose phosphate pathway (PPP) and amino acid (AA)
19 metabolism including glutaminolysis take place in the cytosol. It separates numerous compartments within the cell, with distinct functions and responsibilities. One of these specialized compartments within eukaryotic cells are mitochondria. Mitochondria are thread like structures that are highly adaptable to cellular energy needs. They are the site of the tri-citric acid (TCA) cycle as well as the electron transport chain (ETC) and OXPHOS. Mitochondria are made up of an inner and an outer mitochondrial membrane with the inter-membrane space in between. The inner membrane forms engulfings (cristae) into the inside of the mitochondria, the mitochondrial matrix. Cristae are the site of ETC complexes and energy production in the form of ATP.
To meet the cell’s energy demand, mitochondria possess the ability to adapt their morphology. They can fuse together, a process termed mitochondrial fusion and observed to be required in fatty acid oxidation (FAO) and memory T cell development [62],[63]. During fusion, mitochondrial cristae move further apart and thereby decrease ETC efficiency and energy production in the form of ATP. Additionally, the division of mitochondria, termed mitochondrial fission, has been observed in effector T cells and increases mitochondrial energy production. Fission induces mitochondrial cristae, and with that ETC complexes, to move closer together and thereby allows for a greater efficiency of energy production, specifically, ATP generation [62],[63].
Fusion and fission are also required for the regulation of mitophagy. Mitophagy has first been described by Lemaster and colleagues as the process of degradation of damaged mitochondria [64]. In cellular division, fission processes allow the equal distribution of mitochondria between cells, induce the segregation of damaged mitochondria and initiation of mitophagy [65],[66]. A most recent comprehensive review by Williams and Ding summarizes the complex field of mitophagy and the underlying molecular mechanisms and pathophysiological roles [67]. Briefly, the authors describe nuclear and mitochondrial genes involved in mitophagy processes and the relevance of mitophagy processes in innate and adaptive immunity. E.g., in T cells, mitophagy is essential for cellular differentiation and mitophagy deficiency has been shown to increase mitochondria abundance, toxic reactive oxygen species and with that lead to overall impaired peripheral T cell survival [68]. The relevance of mitophagy with respect to auto-immune diseases requires future investigations.
20 Mitochondria possess their own mitochondrial DNA (mt-DNA) as well as ribosomes for protein synthesis. Mt-DNA is believed to have its origin in the endosymbiotic theory and encodes about 37 genes that are coding for ETC molecules and amino acids [69],[70]. In sexual reproduction, mitochondria are inherited via the egg cell from the mother.
Mitochondria-associated membranes (MAMs) directly connected mitochondria to the endoplasmic reticulum (ER) and the nucleus allowing immediate and efficient communication between the cell structures. Among other molecules, lipids and calcium ions (Ca2+) can be shuttled between the ER and mitochondria, which is essential in energy metabolic homeostasis [71].
An overview of the major metabolic pathways is given in Figure 2. Glycolysis, the PPP, FAO, glutaminolysis, the TCA cycle, the ETC and OXPHOS are shown as the main energy producing pathways within the cell.
Figure 2: Overview of main cellular energy pathways. In energy production pathways, nutrients are taken up from the interstitial fluid outside the cell into the cytosol. Subsequently, they get catabolized via numerous chemical reactions yielding energy-rich ATP, NADH and FADH2
molecules. Glycolysis, PPP, glutaminolysis (all taking place in the cytosol), FAO, ETC and OXPHOS (all occurring in the mitochondria) are the main pathways during which glucose, fatty acids and glutamine are broken down. OXPHOS requires the presence of molecular oxygen (O2). Metabolic
waste products like carbon dioxide (CO2), ethanol and lactic acid are secreted from the cell. AA:
Amino Acids, ATP: Adenosine Triphosphate, ETC: Electron Transport Chain, FADH2: Flavin Adenine
Dinucleotide H2, FAO: Fatty Acid Oxidation, NADH: Nicotinamide Adenine Dinucleotide H,
OXPHOS: Oxidative Phosphorylation, PPP: Pentose Phosphate Pathway, TCA: Tri-citric Acid Cycle. (designed based on [62],[63],[72]–[76])
21
1.3.1.1 Fatty Acid β-oxidation and the Mitochondrial Membrane Protein CPT1a
Fatty acid β-oxidation (short β-oxidation or FAO) describes the catabolism of fatty acids to numerous products that the cell can use for energy production. The starting point of this reaction is the beta carbon of the fatty acid molecule, hence the name β-oxidation. In order for FAO to commence, fatty acids need to be activated and subsequently transferred from the cytosol via the mitochondrial membrane into the mitochondrion. The activation of fatty acids involves an enzyme-mediated reaction in the cytosol during which acetyl-coenzyme A (acetyl-CoA) is bound to the fatty acid. Short-chain fatty acids (between 2 and 6 carbon atoms in size) can diffuse into the mitochondria. However, medium- and long-chain fatty acids (larger than 6 carbon atoms) cannot diffuse into mitochondria and require a specific transporter [72],[74].
The central molecule facilitating this transfer is CPT1a (Carnitine palmitoyltransferase I isoform a), which is located in the mitochondrial membrane. CPT1a is a member of the carnitine/choline acetyltransferase family and located on chromosome eleven [49]. The mitochondrial trans-membrane protein forms homohexamer and homotrimer complexes with Acyl-CoA Synthetase Long Chain Family Member 1 (ACSL1) and Voltage Dependent Anion Channel (VDAC) 1 and has also been found in complexes with VDAC2 and VDAC3 [49].
CPT1a activates fatty acid by conjugating medium- and long-chain fatty acids to carnitine. This allows the conjugated fatty acid acyl-CoA to be shuttled into the mitochondrion where it is transferred back to fatty acid acyl-CoA by CPT2 (Carnitine palmitoyltransferase 2) [76]. Now, FAO can begin yielding acetyl-CoA, NADH (Nicotinamide Adenine Dinucleotide H) and FADH2 (Flavin Adenine Dinucleotide H2). The lipid synthesis
intermediate malonyl-CoA inhibits CPT1a once fatty acid synthesis (FAS) occurs in a nutrient abundant environment. Therefore, the transfer of fatty acids via CPT1a is the rate-limiting step and CPT1a the key regulatory protein in FAO.
Compared to glycolysis, FAO is very efficient and allows the generation of large amounts of ATP from complex fatty acid molecules. For example, the complete FAO of one palmitate molecule can produce more than 100 ATP molecules, while one glucose molecule can only yield up to 36 ATP molecules [79].
22 The generated NADH and FADH2 molecules from FAO are electron donors for the ETC and
finally OXPHOS for the generation of ATP, which is subsequently described in more detail.
1.3.1.2 Electron Transport Chain and Oxidative Phosphorylation
During oxidative phosphorylation (OXPHOS), electrons are transported down an electrochemical proton gradient of the electron transport chain (ETC). The ETC is made up of complex I to complex V and is integrated into the inner mitochondrial membrane [80] [81]. It is a series of redox (reduction and oxidation) reactions performed by numerous enzymes during which electrons are transferred from electron donors to acceptors, allowing the transfer of hydrogen protons (H+) from the mitochondrial matrix to the inter-membrane space. This generates a proton gradient, which is used for ATP synthesis. Importantly, the process of ADP (adenosine diphosphate)-phosphorylation to form energy-rich ATP requires the presence of molecular oxygen (O2)- hence it is termed
OXPHOS. Therefore, the generation of energy via the ETC is a process of aerobic respiration and an oxygen-dependent metabolic pathway.
The five complexes of the ETC (Figure 3) each have distinct properties and complement each other. In complex I, also termed NADH dehydrogenase, two electrons are removed from the electron donor NADH. In addition to NAD+, the redox reaction yields hydrogen
protons (H+), which are pumped into the mitochondrial inter-membrane space. The
reaction also yields electrons, which are transported to the next complex in the ETC, complex II. The function of complex I can be inhibited by e.g. rotenone (Table 2) preventing the electron transfer to complex II and causing an accumulation of electrons in the mitochondrial matrix as well as the inhibition of hydrogen proton transfer into the inter-membrane space.
Complex II (succinate dehydrogenase) is made up of four subunits and the succeeding complex in the ETC. In addition to electrons derived from complex I, complex II receives electrons from electron donors like succinate via its cofactor FADH2. This complex does
not contribute to the proton transfer into the inter-membrane space, however transfers its electrons to the next complex in the ETC:
In complex III (termed bc1 complex (ubiquinol:cytochrome c oxidoreductase), a series of redox reactions yields electrons that are transferred to complex IV via cytochrome c.
23 Additionally, hydrogen protons are pumped into the inter-membrane space further contributing to the build up of the electrochemical proton gradient. Complex III can be inhibited by antimycin A (Table 2).
The succeeding complex in the ETC is complex IV. The transmembrane protein complex is also known as cytochrome c oxidase. It receives electrons from cytochrome c of complex III and transfers them to molecular oxygen, which yields a H2O molecule. During this
process, hydrogen protons are transferred into the inter-membrane space.
The uneven difference in hydrogen proton concentration between the mitochondrial matrix and inter-membrane space is used by ATP-synthase, complex V, of the ETC. It constitutes two regions with numerous subunits and is the final step of the ETC during which ATP-generation occurs from ADP and phosphate. The hydrogen protons are passed via the ATP-synthase into the matrix allowing ATP synthesis to occur. The process of ADP-phosphorylation can be disrupted by the chemical agent FCCP (Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) (Table 2), which is an uncoupling agent transporting hydrogen protons into the mitochondrial matrix, thereby disrupting the proton gradient and with that preventing the process of ATP synthesis.
Figure 3: Display of the mitochondrial electron transport chain. The electron transport chain (ETC) is located at the inner membrane of mitochondria. Electrons are donated to complex I of the ETC and transported in a series of redox reactions to complex II, III, IV and V. This allows the transfer of hydrogen protons (H+) from the mitochondrial matrix to the inter-membrane space
generating a proton gradient between the mitochondrial matrix and the inter-membrane space. This proton gradient is used for ATP synthesis by complex V. ADP: Adenosine diphosphate, ATP: Adenosine triphosphate, Pi: inorganic phosphate, FAD: Flavin Adenine Dinucleotide, FADH2: Flavin
Adenine Dinucleotide H2, H+: Hydrogen proton, H2O; water molecule, NAD: Nicotinamide Adenine
Dinucleotide H, NAD: Nicotinamide Adenine Dinucleotide, redox: reduction and oxidation reaction, O2: oxygen. (designed based on [80])
24
Table 2: Inhibitors of mitochondrial electron transport chain complexes. Table adapted from [82]–[85].
inhibitors mechanism of toxicity
oligomycin inhibits ATP-synthase (complex V) and induces a hyperpolarization of the inter-membrane space
FCCP uncouples mitochondrial inner membrane, inhibits ATP synthesis by allowing free flow of H+ between the inter-membrane space and matrix rotenone inhibits complex I-induced NADH oxidation to NAD
antimycin A complex III inhibition
In summary, the ETC and OXPHOS are a series of efficient and highly specialized reactions within the mitochondrion that require the presence of molecular oxygen and yield tremendous amounts of energy-rich ATP molecules that the cell can use for any metabolic reactions. Predominantly, this energy is received from the oxidation of fatty acids that yield NADH and FADH2 molecules. Furthermore, in addition to FAO, energy can
be generated from glucose molecules in a process termed glycolysis.
1.3.1.3 Glycolysis and the Glucose Transporter GLUT1
Glycolysis takes place in the cytosol and is a key metabolic pathway during which glucose (C6H12O6) is catabolized in consecutive reactions into pyruvate along with numerous
byproducts. The glycolytic pathway begins with the uptake of extracellular glucose from the interstitial fluid, the immediate cellular microenvironment surrounding the cell. Glucose molecules require a transporter in order to cross the cell membrane. One of the main trans-membrane glucose transporter proteins with a high affinity of glucose is Glucose transporter 1 (GLUT1), which is encoded by the gene Scl2a1. It is part of a family of glucose transporters including GLUT1 to GLUT14, each of which expressed in specific body tissues and cells [75].
The intracellular processing of glucose in the glycolytic pathway entails a series of enzymatic reactions yielding pyruvate molecules. Under anaerobic conditions, pyruvate can be processed into lactate and ethanol. Under aerobic conditions, pyruvate is processed to acetyl-CoA and entered into the TCA cycle, which takes place in the mitochondrial matrix. Anaerobic glycolysis can occur even though oxygen is abundantly
25 present in the cell, e.g. during inflammatory processes or in cancer cells (Warburg effect). However, anaerobic glycolysis is less efficient in the generation of energy-rich ATP. The catabolism of one glucose molecule yields two ATPs during anaerobic glycolysis and 36 ATPs if processed via the TCA cycle and ETC respiration [76]. Lactate and CO2 are waste
products that are secreted by the cell and are associated with an increased acidic microenvironment [86].
Nevertheless, there are central benefits of glycolysis, including the generation of the co-factor NADH, which is essential in cellular anabolism. Furthermore, glyco
